Immunoassay devices and materials

ABSTRACT

The present invention comprises a device for detection of the presence of an analyte in a sample of biological fluid through the use of immunochemical ligand-receptor reactions and specially selected, treated, and arranged filter materials.

BACKGROUND OF THE INVENTION

[0001] Various methods for detecting the presence of an analyte in asample of biological fluid through the use of immunochemistry have beendescribed. In the so-called “sandwich” method, for example, a targetanalyte such as an antigen is “sandwiched” between a labeled antibodyand an antibody immobilized onto a solid support. The assay is read byobserving the presence and amount of bound antigen-labeled antibodycomplex. In the competition immunoassay method, antibody bound to asolid surface is contacted with a sample containing an unknown quantityof antigen analyte and with labeled antigen of the same type. The amountof labeled antigen bound on the solid surface is then determined toprovide an indirect measure of the amount of antigen analyte in thesample.

[0002] Because these and other methods discussed below can detect bothantibodies and antigens, they are generally referred to asimmunochemical ligand-receptor assays or simply immunoassays.

[0003] Solid phase immunoassay devices, whether sandwich or competitiontype, provide sensitive detection of an analyte in a biological fluidsample such as blood or urine. Solid phase immunoassay devicesincorporate a solid support to which one member of a ligand-receptorpair, usually an antibody, antigen, or hapten, is bound. Common earlyforms of solid supports were plates, tubes, or beads of polystyrenewhich were well known from the fields of radioimmunoassay and enzymeimmunoassay. More recently, a number of porous materials such as nylon,nitrocellulose, cellulose acetate, glass fibers, and other porouspolymers have been employed as solid supports.

[0004] A number of self-contained immunoassay kits using porousmaterials as solid phase carriers of immunochemical components such asantigens, haptens, or antibodies have been described. These kits areusually dipstick, flow-through, or migratory in design.

[0005] In the more common forms of dipstick assays, as typified by homepregnancy and ovulation detection kits, immunochemical components suchas antibodies are bound to a solid phase. The assay device is “dipped”for incubation into a sample suspected of containing unknown antigenanalyte. Enzyme-labeled antibody is then added, either simultaneously orafter an incubation period. The device next is washed and then insertedinto a second solution containing a substrate for the enzyme. Theenzyme-label, if present, interacts with the substrate, causing theformation of colored products which either deposit as a precipitate ontothe solid phase or produce a visible color change in the substratesolution. Baxter et al., EP-A 0 125 118, disclose such a sandwich typedipstick immunoassay. Kali et al., EP-A 0 282 192, disclose a dipstickdevice for use in competition type assays.

[0006] Flow-through type immunoassay devices were designed to obviatethe need for extensive incubation and cumbersome washing stepsassociated with dipstick assays. Valkirs et al., U.S. Pat. No.4,632,901, disclose a device comprising antibody (specific to a targetantigen analyte) bound to a porous membrane or filter to which is addeda liquid sample. As the liquid flows through the membrane, targetanalyte binds to the antibody. The addition of sample is followed byaddition of labeled antibody. The visual detection of labeled antibodyprovides an indication of the presence of target antigen analyte in thesample.

[0007] Korom et al., EP-A 0 299 359, discloses a variation in theflow-through device in which the labeled antibody is incorporated into amembrane which acts as a reagent delivery system.

[0008] The requirement of multiple addition and washing steps withdipstick and flow-through type immunoassay devices increases thelikelihood that minimally trained personnel and home users will obtainerroneous assay results.

[0009] In migration type assays, a membrane is impregnated with thereagents needed to perform the assay. An analyte detection zone isprovided in which labeled analyte is bound and assay indicia is read.See, for example, Tom et al., U.S. Pat. No. 4,366,241, and Zuk, EP-A 0143 574.

[0010] The sensitivity of migration type assays is frequently reduced,however, by the presence or formation in the sample of undesirable solidcomponents which block the passage of labeled analyte to the detectionzone. Assay sensitivity also declines when migration assay devices areflooded with too much liquid sample.

[0011] Migration assay devices usually incorporate within them reagentswhich have been attached to colored labels, thereby permitting visibledetection of the assay results without addition of further substances.See, for example, Bernstein, U.S. Pat. No. 4,770,853, May et al., WO88/08534, and Ching et al., EP-A 0 299 428.

[0012] Among such labels are gold sol particles such as those describedby Leuvering in U.S. Pat. No. 4,313,734, dye sol particles such asdescribed by Gribnau et al., in U.S. Pat. No. 4,373,932 and May et al.,WO 88/08534, dyed latex such as described by May, supra, Snyder, EP-A 0280 559 and 0 281 327, and dyes encapsulated in liposomes by Campbell etal., U.S. Pat. No. 4,703,017. These colored labels are generally limitedin terms of the immobilization methods which are suitable. Moreover,they require a relatively large amount of ligand molecule and caninvolve expensive reagents, thereby adding to the cost.

DETAILED DESCRIPTION

[0013] The present invention comprises a device for detection of thepresence of an analyte in a sample of biological fluid through the useof immunochemical ligand-receptor reactions and specially selected,treated, and arranged filter materials. The nature of the invention willbe apparent from the following description and from the accompanyingdrawings in which;

[0014]FIG. 1 is a cross sectional view of a typical unidirectional assaydevice;

[0015]FIG. 2 is a cross sectional view of a second embodiment of aunidirectional assay device;

[0016]FIG. 3 is a cross sectional view of a typical bidirectionaldevice;

[0017]FIG. 4 is a perspective view of an assay device as depicted inFIG. 2 (shown in partial phantom lines) encased in a plastic enclosurewith a single aperture;

[0018]FIG. 5 is a top view of a multidirectional assay device; and

[0019]FIG. 6 is a perspective view of an assay device as depicted inFIG. 2 (shown in partial phantom lines) encased in a plastic enclosurewith 2 apertures.

[0020] Referring now to FIG. 1, reservoir pad 10, filter element 12, andwicking membrane 16, which contains immobilized substance defined inassay indicia zone 18, are disposed on base 20. Reservoir pad 10 hassufficient porosity and volume to receive and contain a liquid sample onwhich the assay is to be performed. Filter element 12 is adjacent to andcontiguous across a relatively small surface of reservoir pad 10relative to the volume of pad 10 so as to meter the passage of theliquid sample emerging from reservoir pad 10 into filter element 12.

[0021] Disposed in defined zone 18 of wicking membrane 16 is animmobilized substance which is operable to bind any specific ligandreceptor complexes contained in the sample passing through filterelement 12.

[0022] In this embodiment, a reagent operable to produce a specificligand receptor complex is added to the sample where it will react toform the complex (assuming it contains the appropriate analyte) and thesample then brought into contact with reservoir pad 10. The samplemigrates through filter element 12 where any unwanted components whichmay be present in the sample become trapped, and on into wickingmembrane 16. Labeled analyte if present binds to assay indicia zone 18producing a visibly detectable signal.

[0023] In the embodiment shown in FIG. 2, there is present, in additionto reservoir pad 10, filter element 12, and wicking membrane 16, secondfilter element 14 which is disposed on base 20. Reservoir pad 10 hassufficient porosity and volume to receive and contain a liquid sample onwhich the assay is to be performed. First filter element 12 is adjacentto and contiguous across a relatively small surface of reservoir pad 10relative to the volume of pad 10 so as to meter the passage of theliquid sample emerging from reservoir pad 10 into first filter element12. In this embodiment, a reagent operable to produce a specific ligandreceptor complex is uniformly impregnated throughout first filterelement 12. As liquid sample emerges from reservoir pad 10, it comes incontact with the reagent impregnated in first filter element 12 where itwill react to form the specific ligand receptor complex or complexes(assuming the sample contains the appropriate analyte or analytes). Theuse of the first filter element as a reagent delivery system obviatesthe need for a separate reagent addition step.

[0024] Second filter element 14, adjacent to first filter element 12 anddistal to reservoir pad 10, is operable to permit passage of anyspecific ligand receptor complexes contained or formed in the liquidsample but to impede passage of larger components contained therein,which components may be either present in the original sample orthereafter formed, for example in first filter element 12.

[0025] Wicking membrane 16 is adjacent to second filter element 14 anddistal to first filter element 12. Wicking membrane 16 has sufficientporosity and volume to absorb a substantial proportion of the samplereceived in reservoir pad 10 after passage through first filter element12 and second filter element 14.

[0026] Disposed in defined zone 18 of wicking membrane 16 is animmobilized substance which is operable to bind any specific ligandreceptor complexes formed and contained in the sample passing throughfirst filter element 12 and second filter element 14.

[0027] In use, a liquid sample is applied to reservoir pad 10 of thedevice shown in FIG. 2. The sample migrates through first filter element12, wherein target analytes, if present in the sample, bind to labeledreagent. The sample continues its migration through second filterelement 14 wherein any unwanted components which may be present in thesample become trapped, and on into wicking membrane 16. Labeled analyteif present then binds to assay indicia zone 18 producing a visiblydetectable signal.

[0028] Alternatively, immunstatus assays can be performed by applying asample to second filter element 14 of the device shown in FIG. 2. Abuffer solution is then applied to reservoir pad 10, the solutionmigrates through first filter element 12 reconstituting labeled reagentstherein. The solution and reagents migrate through second filter element14 wherein target analyte if present bind to the labeled reagents, andon into wicking membrane 16. Labeled analyte if present then binds toassay indicia zone 18 producing a visibly detectable signal.

[0029] Referring to FIG. 3, common reservoir pad 110, first filterelements 112A and 112B, second filter elements 114A and 114B, andwicking membranes 116A and 116B, which contain immobilized substancedisposed in defined assay indicia zones 118A and 118B, are disposed onbase 120. Common reservoir pad 110 has sufficient porosity and volume toreceive and contain a liquid sample on which the assay is to beperformed. First filter elements 112A and 112B are adjacent to andcontiguous across a relatively small surface of common reservoir pad 110with respect to the volume of pad 110 in order to meter the passage ofthe liquid sample from reservoir pad 110 to first filter elements 112Aand 112B. Reagent operable to produce a specific ligand receptor complexis uniformly impregnated throughout first filter elements 112A and 112B.Second filter elements 114A and 114B are adjacent to first filterelements 112A and 112B respectively and distal to common reservoir pad110, and are operable to permit passage of any specific ligand receptorcomplexes formed in the liquid sample but impede passage of largercomponents contained therein. Wicking membranes 116A and 116B havesufficient porosity and volume to absorb a substantial proportion of thesample received in common reservoir pad 110. Immobilized substance inzones 118A and 118B is operable to bind any specific ligand receptorcomplexes formed.

[0030] In use, a liquid sample is applied to common reservoir pad 110,the sample migrates through first filter elements 112A and 112B, whereintarget analytes, if present in the sample, bind to the labeled reagentsimpregnated therein, through second filter elements 114A and 114Bwherein any undesirable components of the fluid sample are trapped, andon into wicking membranes 116A and 116B. Target labeled analytes ifpresent bind to assay indicia zones 118A and 118B. Thus the embodimentof FIG. 3 permits the simultaneous and independent assay of two analytesor two parallel assays for the same analyte, in each case using a singlesample.

[0031] Referring to FIG. 4, an assay device such as that shown in FIG. 2is enclosed in casing 222. Casing 222 has aperture 224 situated directlyover reservoir pad 210 and viewing window 226 situated directly overassay indicia zone 218 and assay indicia control zone 228. Window 226can be a simple aperture or can comprise a clear material which protectszones 218 and 228 but permits visual inspection. Liquid sample is addedthrough aperture 224 and is absorbed by reservoir pad 210. It thenmigrates through first filter element 212 carrying the appropriatelabeled reagents through second filter element 214, in which anyunwanted components of the sample are trapped, and on into wickingmembrane 216 in which labeled analyte, if present, binds to assayindicia zone 218. Unbound labeled reagent binds to assay indicia controlzone 228. Both indicia zones 218 and 228 can be visualized throughviewing window 226.

[0032] Referring to FIG. 5, base 320 which is fabricated from a moistureresistant material such as plastic is segmented into a plurality of likeregions 311A, 311B, 311C, 311D, 311E, and 311F by dividers 322. Eachregion comprising a first filter element 312, a second filter element314, and wicking membrane 316 all disposed in base 320 between dividers322. The same or different reagent can be deposited in each first filterelement 312, permitting either parallel tests for the same analyte or aplurality of different assays on the same sample. Each wicking membrane316 contains an immobilized substance, as appropriate for the reagent inthe first filter element associated with its corresponding wickingmembrane, deposited in defined assay indicia zone 318. Common reservoirpad 310 in which the sample is deposited is centrally located withrespect to the plurality of regions.

[0033] In use, a fluid sample placed on reservoir pad 310 migratessimultaneously through first filter elements 312 in which targetanalytes, if present, bind to labeled antibodies. The sample then passesthrough second filter element 314 and into wicking membranes 316 inwhich labeled target analytes, if present, bind to the correspondingimmobilized substance in assay indicia zone 318, producing a visiblydetectable signal.

[0034] The embodiment shown in FIG. 6 can be used in immunstatus assays.An assay device such as that shown in FIG. 2 is enclosed in casing 522.Casing 522 has first aperture 524 situated directly over reservoir pad510, second aperture 530 situated directly over second filter element,and viewing window 526 situated directly over assay indicia zone 518 andassay indicia control zone 528. Window 526 can be a simple aperture orcan comprise a clear material which protects zones 518 and 528 butpermits visual inspection. In use, a sample (as for example serum) isapplied directly onto second filter element 514 through second aperture530. A buffer solution is then applied to reservoir pad 510 throughfirst aperture 524, the solution migrates through first filter element512 reconstituting labeled reagents therein. The solution and reagentsmigrate through second filter element 514 wherein target analyte ifpresent bind to the labeled reagents, and on into wicking membrane 516.Labeled analyte, if present, then binds to assay indicia zone 518producing a visibly detectable signal. Unbound labeled reagent binds toassay indicia control zone 528. Both indicia zones 518 and 528 can bevisualized through viewing window 526.

[0035] Regardless of the configuration, the filter elements and padswill abut or overlap one another so as to define an interface forpassage of the sample. It has been found advantageous if the wickingmembrane has a high area:thickness ratio whereas the filter elementadjacent thereto has a relatively lower area:thickness ratio. To insurean adequate interface, it thus is advantageous to place the adjacentfilter element in overlapping relation to the wicking membrane.

[0036] By incorporating at least one filter element before the assayindicia zone, an increase in sensitivity is achieved as compared toprevious migration type assays. The filter, which preferably has beentreated to reduce any inherent hydrophobicity, traps unwanted componentsin the fluid sample and allows unimpeded passage of labeled analyte.Thus, a proportionately greater amount of analyte binds to the assayindicia zone, and more accurate assay results are achieved.

[0037] Additionally by selecting a membrane with the appropriate textureand pore size, the second filter element can act as a controlled celllysing system. For example in an immunstatus assay performed on a sampleof whole blood it is advantageous to select as the second filter elementa membrane which would maintain the integrity of whole blood cells whileserum migrates through. This prevents the discoloration associated withblood cell lysis from spreading into the assay indicia zone.

[0038] When the device is used to perform an immunstatus assay, thisadditional filter element can also operate to receive samples directly.Generally, these assays are performed on samples of whole blood or serumwhich are spotted directly onto the filter. This is followed by theapplication of a buffer solution to the reservoir pad. Typical buffersolutions include, but are not limited to, phosphate buffer solution,saline, Tris-HCl, and water. Examples of antibodies which may bedetected in this fashion include, but are not limited to, AcquiredImmune Deficiency Syndrome, Rubella, Hepatitis, and Lymes.

[0039] Another source of assay sensitivity decline, sample flooding,also is avoided by the incorporation of the reservoir pad in the device.Thus the reservoir pad can hold a large quantity of sample which then ismetered through the device as a result of the effective interface in thesubsequent zones. This aspect of the invention makes it particularlysuitable, for example, for home use where the device may be placed in astream of urine without the need to measure the quantity of the sampleapplied to the device.

[0040] The reservoir pad, first filter element, second filter element,and wicking membrane are fashioned from any number of filter materials.Typical filter materials for use in the reservoir pad include, but arenot limited to, low protein binding materials such as cellulose,polyesters, polyurethanes, and fiberglass with pore sizes in the rangeof 0.45-60 μm. Typical materials for use in the first filter elementinclude, but are not limited to, cellulosic materials (e.g., Whatmanpaper ET31) or fiberglass with pore sizes in the range of 0.45-60 μm.Typical materials for use in the second filter element are hydrophilicmaterials which include, but are not limited to, polyurethane,polyacetate, cellulose, fiberglass, and nylon with pore sizes in therange of 0.45-60 μm. Typical materials for use in the wicking membraneinclude, but are not limited to, nylon, cellulose, polysulfone,polyvinylidene difluoride, cellulose acetate, polyurethane, fiberglass,and nitrocellulose.

[0041] The entire array of pads and membranes is attached to a solidmaterial which provides support and unity for the device. This supportcan be constructed from a thin sheet of glass or plastic which has beencut to a size appropriate to include entire assay contents whileproviding convenience to the assay user.

[0042] Another embodiment of the present invention permits the detectionof multiple analytes in a single fluid sample by the presence of morethan one specific type of labeled reagent and the same number of typesof corresponding immobilized reagent. The device can be set upunidirectionally with multiple labeled reagents impregnated throughoutone first filter element and multiple corresponding immobilizedsubstances defined in several assay indicia zones on the wickingmembrane. In the bidirectional or multidirectional embodiment, more thanone set of components such as first filter element, second filterelement, and wicking membrane are associated with a common reservoir.

[0043] In each case, reagents which are incorporated in the first filterelement are either dried or lyophilized onto or into the element inorder to allow their reconstitution upon contact with a liquid sample.Other reagents which are useful to enhance the specificity or increasethe number of ligand receptor complexes created and bound and henceincrease the sensitivity of the assay device, also can be included inthe filter pad, or in the first of two filter pads which also willcontain the reagent. These auxiliary reagents include, but are notlimited to, buffers, detergents, and anticoagulants.

[0044] By including an additional assay indicia zone to serve as acontrol on the wicking membrane adjacent to the first zone and distal tothe second filter element, the assay device presents an internal monitorwhich indicates whether liquid sample has migrated throughout the entiredevice. Assay indicia control zones generally employ immobilizedantibodies (such as anti-immunoglobins) to the labeled reagents whichhave been added to the analyte or incorporated into the first filterelement. With reference to the embodiment of FIG. 2, for example, aliquid sample migrates through the first filter element where itreconstitutes the labeled reagent and carries it to the wickingmembrane. Labeled reagent which is not bound to target analyte binds tothe control assay indicia zone, creating a visibly detectable indicationof test completion.

[0045] The entire assay device, whether constructed as unidirectional,bidirectional, or multidirectional, can be encased in a liquidimpervious plastic. This encasing normally has an aperture over thereservoir filter in order to receive sample. The entire encasement canbe transparent or a portion over the analyte detection zone can betransparent in order to observe assay results. Devices encased inplastic are especially useful and convenient for use in home diagnostickits but other materials such as treated paper also can be employed.

[0046] Additionally, the upper surface surrounding the aperture can becurved and extended downwards so as to form a cup-like receptacle whichterminates at and firmly engages a portion of the reservoir pad. In thisway the amount of sample introduced into the device is metered and thesample cannot bypass any components of the device.

[0047] The present invention can be used with either competition assaysor sandwich assays. In competition assays, an additional inclusion of alabeled antigen (which is the same as the target antigen) is includedeither separately or as part of the first filter element. This labeledantigen competes with antigen from the sample for binding on thedetection zone.

[0048] The diagnostic devices and methods described in the presentinvention can be used in any ligand-receptor reactions and areespecially suited for those reactions having immunochemical componentssuch as antibodies, antigens, and haptens. In those assays in which thedetection of ligand containing molecules such as antigens or haptens isdesired, both the labeled reagent and substance immobilized in the assayindicia zone are ligand binding molecules. More specifically, when theligands are antigens, both the labeled reagents and immobilized reagentswill be antibodies.

[0049] The antibodies can be either monoclonal or polyclonal, themethods of producing which are well known in the art. It is preferableto use labeled monoclonal antibodies in the first filter element andpolyclonal antibodies in the assay indicia zone in order to achievemaximum binding. However, any combination of monoclonal-polyclonalantibodies can be employed. In the case of home pregnancy and ovulationpredictor kits, antibodies are made to human chorionic gonadotrophin andluteinizing hormones respectively and are incorporated into the device.

[0050] In those instances in which detection of a ligand bindingmolecule such as an antibody is desired, labeled mouse antihumanimmunoglobin G is employed as the reagent which, as noted, can beincorporated in the first filter element and antigen specific to thetarget antibody is immobilized in the assay indicia zone. Any natural orsynthetic antigen can be employed as well as polypeptide chains whichhave antigenic activity. Examples of antigens which can be immobilizedonto the device include, but are not limited to, Rubella, Lymes,Acquired Immune Deficiency Syndrome, Hepatitis, Toxoplasmosis,cytomegalovirus, and Epstein Barr Virus.

[0051] In assays where the simultaneous detection of more than oneantibody in a single sample is desired, since the same labelledantihuman immunoglobin (reagent) recognizes and binds to all humanantibodies present in the sample, it is only necessary to incorporateantigens for each target antibody in distinct indicia zones on thewicking membrane.

[0052] The labels employed in the device can be either direct orindirect. Direct labels are preferred in that they require no additionalsteps in order to visualize assay results. Examples of direct labelsinclude but are not limited to, metal sols, dye sols, particulate latex,color indicators, colored matter contained in liposomes, and nonmetalsols such as a carbon sol.

[0053] In a further aspect, the invention relates to one immunochemicallabel which is particularly well-suited for use in the foregoing devicebut which can be used in other immunological assays as well, inparticular, an immunochemical label in which to an immunological ligandor ligand binding molecules is linked directly or indirectly to thesurface of finely particulate carbon black.

[0054] The immunological label can be diagrammatically depicted as C-Lor C-X:L in which C is the finely particulate carbon black, “-”represents an adsorption linkage, L is a component containing a ligand-or ligand binding unit, X is a linking agent, and “:” represents acovalent bond.

[0055] L can consist of only the ligand- or ligand binding unit, inwhich case it is adsorbed directly onto the carbon. Alternatively, theligand- or ligand binding unit can be bound to a bridging member eithercovalently or immunologically (herein designated by “*”) For example,the ligand- or ligand binding unit can be covalently bound to a linkingagent such as glutaraldehyde which in turn is covalently bound to aproteinaceous bridging member such as bovine serum albumin (BSA) whichin turn is adsorbed on the carbon. Likewise, avidin or streptoavidin canbe linked through biotin to the ligand- or ligand binding molecule andthe avidin or streptoavidin adsorbed on the carbon particles.Alternatively a primary antibody, serving as the ligand- or ligandbinding unit is immunologically bound to a secondary antibody and thesecondary antibody is adsorbed to the carbon particles. Typicalstructures of the C-L embodiment thus include:

[0056] C-{ligand},

[0057] C-{ligand binding molecule},

[0058] C-{protein:X:ligand},

[0059] C-{protein:X:ligand binding molecule},

[0060] C-{2·°Ab*1·°Ab}, and

[0061] C-{protein:X:2·°Ab*1·°Ab}.

[0062] In a second embodiment, a linking agent Y is both adsorbed on thecarbon particle and covalently bound to the ligand- or ligand bindingunit to form a label of the general formula C-Y:L. The linking agent Ycan be a single molecular species, Y′, as more fully discussed below, orcan be a composite such as linking agent:protein:linking agent:

[0063] C-Y′:{ligand},

[0064] C-Y′:{ligand binding molecule},

[0065] C-Y′:protein:X:{ligand}, and

[0066] C-Y′:protein:X:{ligand binding molecule}.

[0067] It will be noted that the principal difference between the twoembodiments is that in the first embodiment, a ligand, ligand bindingmolecule, or protein (such as an antibody, bovine serum albumin, oravidin) is adsorbed on the carbon particles whereas in the secondembodiment, a member of a particular class of organic compounds servingas a linking agent is adsorbed on the carbon particles and covalentlybound to a ligand, ligand binding molecule, or protein.

[0068] The foregoing carbon sols can be prepared by a number of methods.The ligand and ligand binding molecules can be simply added to asuspension of the carbon particles to produce C-{ligand} and C-{ligandbinding molecule} structures. In instances in which the ligand or ligandbinding molecule is bound indirectly, the full, non-carbon particlestructure such as {protein:X:ligand}, {protein:X:ligand bindingmolecule}, {2·°Ab*1·°Ab}, or {protein:X:2·°Ab*1·°Ab} can be prepared andthen added to a suspension of the carbon particles for adsorption.Alternatively, a terminal portion of the non-carbon particle structurefirst can be adsorbed on the carbon particles and the remainder of thenon-carbon particle structure then introduced chemically. For example, aprotein such as bovine serum albumin, avidin, or streptoavidin can beadsorbed on the carbon particles and then linked, using for exampleglutaraldehyde for bovine serum albumin or biotin for avidin orstreptoavidin, to the ligand or ligand binding molecule. Similarly, a2·°antibody can be adsorbed on the carbon particles and a 1·°antibodythen joined immunologically.

[0069] Linking reagent Y′ suitable for covalently-linking ligand andligand binding molecules such as haptens, antigens, or antibodies, orfor covalently-linking protein bridging groups, include imides, azides,isothiocyanates, imidoesters, and dialdehydes, as for example,maleimide, succinimide, phenylazide, glutaraldehyde,N-hydroxysuccinimide ester, phenylisothiocyanate,4,4′-diisothiocyanostilbene-2,2′-disulfonic acid,4-N,N-dimethylaminoazobenzene-4′-isothiocyanate, flouresceinisothiocyanate, rhodamineisothiocyanate, and the like.

[0070] As in the case of the first embodiment, the complete non-carbonparticle structure, prepared by reacting the ligand (or ligand bindingmolecule), any bridging protein, and linking agent, can be adsorbed onthe surface of the finely particulate carbon black. Alternatively, thelinking reagent alone first can be adsorbed on the finely particulatecarbon black and then covalently bound to the ligand, ligand bindingmolecule, and/or bridging protein.

[0071] In any of the above procedures, it generally is desirable to adda suspending adjuvant to the aqueous suspension of the finelyparticulate carbon black, as for example a polyalkylene glycol orpolysaccharide. As will be seen below, similar substances subsequentlyare added as a protective agent after linking the immunological ligandor ligand binding molecules to the finely particulate carbon black. Theamount added at this stage thus is relatively small, generally beingthat sufficient merely to assist in the suspension of the carbonparticles.

[0072] The linking reagent then is allowed to both (i) react covalentlywith the immunological ligand or ligand binding molecules and (ii) beadsorbed on finely particulate carbon black, either simultaneously orsequentially. While dependent on the particular linking reagent, thelinking reaction generally is conducted over several hours at pH valuesof from about 7.0 to about 9.5.

[0073] A variety of commercially available finely particulate carbonblack materials can be used such as Monarch 1,000, 120, or 880, VulcanXC72 or XC72R, or Regal 250R or 500R. The suitability of any particularsource can be readily determined by homogenating the material in bufferand measuring the optical density.

[0074] Preferably, the finely particulate carbon black with the ligandor ligand binding molecule bound covalently or passively is treated witha polyalkylene glycol or polysaccharide protective agent to minimizehydrophobicity and maximize dispersability. Suitable materials for suchcoating are polyethylene glycols having a molecular weight of from about100 to about 20,000, preferably from about 5,000 to about 12,000, andprotective polysaccharides such as dextran having a molecular weight offrom about 10,000 to about 500,000, preferably from-about 10,000 toabout 50,000. This coating can be readily achieved by contacting thelinked carbon black with a 0.5% to 5% weight/volume aqueous solution ofthe polyethylene glycol or dextran.

[0075] In a further embodiment, the immunochemical label is treated withat least one biologically acceptable ionic or nonionic surfactant, suchas long chain alkyl trimethylammonium salt, sodium deoxycholate,Tritons, Tweens, etc., in a concentration range of from about 0.01 toabout 0.5%. After each such treatment, of which there can be several,with the same or different types of detergent, the immunochemical labelis washed to remove excess detergent.

[0076] The resulting immunochemical label then can be suspended in anaqueous media. Such aqueous suspensions of the immunochemical label areparticularly useful for the fabrication of immunoassay devices, boththose of the present invention and those of other structures. Preferablythe aqueous suspension includes at least one buffer in order to providea pK_(a) at which the labeled immunological ligand or ligand bindingmolecule is stable; e.g., within the range of from about 6 to about 9and preferably from about 6.5 to about 8.5.

[0077] The following examples will serve to further typify the nature ofthis invention but should not be construed as a limitation on the scopethereof, which is defined solely by the appended claims.

Sensitivity Procedure

[0078] Sensitivities are determined in the following examples bypreparing standard solutions of human chorionic gonadotropin inconcentrations of 25 mIU/ml, 50 mIU/ml, 75 mIU/ml, and 100 mIU/ml.Samples (0.15-0.20 ml) of the standard are applied to the assay deviceand sensitivity determined by the ability of the device to detect agiven concentration of human chorionic gonadotropin.

EXAMPLE 1

[0079] A. Preparation of Label. Gold sol particles are dissolved in 750ml of distilled water which is then brought to a boil. Hydroauric acid(70 to 75 mg) is added, and boiling is continued for 5 minutes. Sodiumcitrate (80 mg) dissolved in 10 ml of distilled water is poured into thegold solution and the solution boiled for another 5 minutes. Afterallowing the solution to cool to ambient temperature, its pH is adjustedto a range close to the isoelectric point for the monoclonal antibody(determined using gel electrophoresis) made against human chorionicgonadotropic hormone. Twenty milligrams of the monoclonal antibody areadded to the solution which is stirred for 2 hours at ambienttemperature. Seven hundred and fifty milligrams of bovine serum albuminare added, and the solution is continuously stirred for approximately 12hours at ambient temperature. Colloidal gold-monoclonal antibodyconjugate is recovered by centrifugation at 10,000 RPM in a GSA rotorfor 20 minutes, discarding the supernatant and suspending the resultantpellet in 30 ml of 1% bovine serum albumin in phosphate buffer solution(pH 7.4). The suspension is then spun down at 16,000 RPM for 15 minutesin a Sorvall SS-34 rotor. The supernatant once again is discarded andthe pellet suspended in 15 ml of 1% bovine serum albumin. After a briefsonication, the suspension is filtered through a 0.2 μm filter.

[0080] B. Preparation of Device. A sample of preactivated nylon membrane(Pall Immunodyne) with a pore size of 5 μm is cut to 180 mm×25 mm sizeand attached to the bottom of a thin plastic plate (100 mm×180 mm) asthe wicking membrane. An assay indicia zone of immobilized antibody isdefined on the membrane by spraying 36 μl of a solution of 3 mg/ml sheepanti-human chorionic gonadotrophin (hCG) antibody in 0.1 M sodiumphosphate buffer (pH 7.6) and 5% sucrose in a line approximately 1.5 cmfrom the bottom using a Camag Linomat IV. After spraying, the membraneis dried at 37° C. for 30 minutes and then treated with a solution of 2%nonfat dry milk (Carnation) and 2% sucrose in 0.1 M sodium phosphatebuffer. The membrane then is washed with 2% sucrose in 0.1 sodiumphosphate and allowed to stand at ambient temperature for approximately12 hours for further drying. The base and wicking membrane can be storedin a desiccator until further processed.

[0081] Two cellulose membranes (Whatman ET31) are pretreated with asolution of 0.1 M sodium phosphate buffer (pH 7.4), 0.1% bovine serumalbumin, 0.5% nonfat dry milk, 2% sucrose, and 0.05% sodium azide andthen incubated for 30 minutes at ambient temperature.

[0082] The second filter element and a reservoir pad are prepared bydrying the 2 pretreated cellulose membranes in a vacuum desiccator for 1hour at ambient temperature.

[0083] The first filter element is prepared by incubating a rectangularpiece of cellulose membrane (Schleicher & Schuell) measuring 5 mm×180 mmat ambient temperature for 30 minutes in a solution of colloidalgold-monoclonal antihuman chorionic gonadotropin antibody conjugate in a0.1M sodium phosphate buffer (pH 7.6) and 5% sucrose. The membrane thenis placed on a glass plate and heat dried at 36° C. under a constantvacuum in a lyophilizer and stored dry in a desiccator until use.

[0084] The first filter element is attached adjacent to the secondfilter element and the second filter element is attached to the plasticbase adjacent to the wicking membrane. Finally the reservoir pad isattached adjacent to the first filter element. The plastic plate then iscut into a plurality of strips 100 mm in length and 7.5 mm in width sothat each contains a linear array of reservoir pad, first filterelement, second filter element and wicking membrane.

EXAMPLE 2

[0085] The same procedure as Example 1B is followed except that thematerial used for the wicking membrane is a cellulose membrane(Schleicher & Schuell) having a pore size of 12 μm. After line-sprayingwith antibody, the membrane is placed in a desiccator for 24 hours toinsure maximum antibody-membrane binding. The membrane then is incubatedin a blocking buffer of 1% bovine serum albumin, 0.5% nonfat dry milk,5% trehalose, 0.05% Tween 20, and 0.05% sodium azide in 0.1M boratebuffer in a pH range of 8.5 to 9.0. The blocked membrane is dried againin a vacuum desiccator for 1 hour and stored in a regular desiccatoruntil incorporation into the assay device.

EXAMPLE 3

[0086] A strip is prepared according to the procedure in Example 1B.

[0087] When a stream of urine is applied to the reservoir pad adetectable signal begins to appear in the assay indicia zone after about3 minutes. The assay sensitivity is about 50 mIU/ml.

EXAMPLE 4

[0088] A strip is prepared according to the procedure of Example 1Bomitting the first filter element and reservoir pad. The strip isinserted in a tube of 100 μl female urine which contains 10 μl ofcolloidal gold-monoclonal anti-human chorionic gonadotropin antibodyconjugate prepared in accordance with Example 1. As the liquid migratesalong the strip, a detectable signal appears in the assay indicia zonein about 2 minutes which becomes stronger by the time the liquid reachedthe end of the strip (about 4 minutes). The sensitivity of this assayprocedure to human chorionic gonadotropin present is 25 mIU/ml.

EXAMPLE 5

[0089] A test strip is prepared substantially in accordance with theprocedure in Example 1B but treating the first filter element with aanti-human chorionic gonadotropin antibody labeled as follows. A 10%suspension (0.1 ml) of commercially available colored polystyrene latexparticles ranging in size from 0.1 to 0.3 μm is washed three times withdistilled water by microfuge centrifugation. The final pellet issuspended in 2 ml of 0.1M glycine hydrochloride buffer containing 1 mgof bovine serum albumin. After approximately 12 hours of incubation atambient temperature on a rocker, the latex suspension is washed 3 timeswith 0.1M sodium phosphate buffer (pH 6.8) to remove excess bovine serumalbumin. The resultant suspension is brought to 2 ml with the samephosphate buffer, and 25% glutaraldehyde is added to a finalconcentration of 1%. The sample is incubated for 3 hours at ambienttemperature and washed three more times with the same buffer. Onehundred micrograms of anti-human chorionic gonadotropin antibody areadded to 2 ml of the latex suspension and incubated for another 3 hoursat ambient temperature. Glycine then is added to a final concentrationof 2%. After an additional hour of incubation, the latex suspension iswashed 3 times with the same buffer and suspended in the same buffercontaining 2% bovine serum albumin. The suspension is briefly sonicatedand then stored at 4° C. until use.

EXAMPLE 6

[0090] A strip is prepared according to the procedure of Example 1Bomitting the first filter element and the reservoir pad. The test stripthen is inserted into a test tube containing 5 μl anti-human chorionicgonadotropin antibodies labeled in accordance with the procedure inExample 5 in 100 μl of female urine. As the liquid migrates along thestrip, a detectable signal appears in the assay indicia zone in about 2minutes, which signal becomes stronger by the time the liquid reachedthe end of the strip (about 4 minutes). The sensitivity of this assayprocedure to human chorionic gonadotropin is 25 mIU/ml.

EXAMPLE 7

[0091] Ten milligrams of Vulcan XC72 carbon particles are homogenized in2 ml of 20 mM Tris-hydrochloride buffer (pH 6.8) containing 40 mM sodiumchloride and 2% dextran 9,400. After 2 hours incubation at ambienttemperature, a solution of 5 mg of flourescein isothiocyanate in 1 ml ofTris-hydrochloride buffer is added to the solution. The mixture isbriefly sonicated and incubated for approximately 12 hours at ambienttemperature. After incubation, 20 ml of 0.1 M sodium phosphate buffer(pH 7.6) in 0.1 M sodium chloride are added to the carbon solution whichthen is centrifuged at 4° C. at 15,000 RPM. This step is repeated threetimes and the resultant pellet suspended in 20 ml of phosphate buffer.After brief sonication, 3 mg of a monoclonal antibody made against humanchorionic gonadotropin are added to the suspension, and the mixtureincubated for 6 hours at ambient temperature. The mixture then iscentrifuged three times at 15,000 RPM to remove unreacted antibody. Thefinal pellet is suspended in 20 ml of 0.1 M Hepes buffer (pH 7.5)containing 1% bovine serum albumin, 5% sucrose, 0.1M sodium chloride,and 0.05% sodium azide. Cetyltrimethyl ammonium bromide is added until afinal concentration of 0.025% is achieved. This then is incubated for 30minutes and centrifuged at 15,000 RPM. The resultant pellet is suspendedin 20 ml of 0.1 M Hepes buffer (pH 7.5) containing 1% bovine serumalbumin, 5% sucrose, 0.1 M sodium chloride, and 0.05% sodium azide,sonicated briefly, and diluted with sodium deoxylate to a finalconcentration of 0.1%, after which it is incubated for 30 minutes atambient temperature and recentrifuged. The pellet again is suspended in20 ml of 0.1 M Hepes buffer (pH 7.5) containing 1% bovine serum albumin,5% sucrose, 0.1M sodium chloride, and 0.05% sodium azide and sonicatedbriefly.

[0092] This carbon sol is introduced in place of colloidal gold on thefirst filter element of test strips prepared according to Example 1Bomitting the reservoir pad. The test strips are dried in a vacuum drierfor about an hour and stored in a desiccator at ambient temperatureuntil use.

[0093] To carry out human chorionic gonadotropin or luteinizing hormoneassays, 100 μl of urine sample is dispensed in a culture tube and thestrip is then inserted into the tube Upon contact with the urine sample,the carbon particle-antibody conjugates immediately become solubilizedand migrate toward the wicking membrane. A positive test corresponds toan intense color of the carbon black particles concentrated in theindicia. The detection limit is about 25 mIU/ml in both the humanchorionic gonadotropin and luteinizing hormone assays.

EXAMPLE 8

[0094] A strip is prepared according to the procedure of Example 1Bomitting the first filter element and the reservoir pad. The test stripthen is inserted into a test tube containing the carbon sol labeledantibody (5 μl), and a urine sample containing human chorionicgonadotropin (100 μl) which are mixed thoroughly. A detectable signalbegins to appear after about 1 minute. The sensitivity of this assaymeasured about 25 mIU/ml.

EXAMPLE 9

[0095] Carbon sol reagents coated with monoclonal antibodies againsthuman chorionic gonadotropin or luteinizing hormone (5 μl per tube) arelyophilized. The tubes can be stored in a desiccator at ambienttemperature until use.

[0096] To conduct human chorionic gonadotropin or luteinizing hormoneassays, 100 μl of urine sample are dispersed in a culture tubecontaining the dried or lyophilized carbon sol. The carbon reagentimmediately goes into solution upon the contact with a urine sample. Atest strip prepared as in Example 1B but without the first filterelement and reservoir pad and on which has been sprayed a line of sheepanti-whole human chorionic gonadotropin antibody (3 μg per strip) as theindicia then is inserted into the tube. When the migrating samplemixture reaches the indicia, a black band begins to appear if the urinesample contained human chorionic gonadotropin or luteinizing hormone.The sensitivity of the assays using the dried or lyophilized carbonreagent is about 25 mIU/ml in both cases. The dried or lyophilizedcarbon reagent remains active, showing the same sensitivity followingstorage for over a year at ambient temperature.

EXAMPLE 10

[0097] An assay device was prepared according to Example 1B eliminatingthe first filter element and reservoir pad and using 1 mg/ml ofanti-thyroxine antibody as the line spray on the wicking membrane.

[0098] Upon insertion in a mixture of 5μ of carbon sol linked tothyroxine (see Example 21) and 100 μl of serum (competitive assay), thecontrol band begins to appear in about two minutes. At thyroxine(unlabeled) levels in the serum sample higher than about 60 ng/ml, noband formation occurs (faint band appears at 59 ng/ml). In contrast, toproduce a band as strong as the control band in the absence of thyroxinein the serum sample, less than 10 ng/ml of thyroxine is required.

EXAMPLE 11

[0099] An assay device was prepared according to Example 1B eliminatingthe first filter element and reservoir pad and using 2 mg/ml ofcommercially available Lymes antigen (OEM Concepts) as the line spray onthe wicking membrane.

[0100] When the reservoir end of the device is dipped into a mixture of100 μl of human serum and 5 μl of carbon particles labeled withflourescein isothiocyanate-conjugated goat anti-human Immunoglobin G(see Example 21), a detectable signal appears in about 4 minutes if thesample is seropositive.

EXAMPLE 12

[0101] An assay device was prepared according to Example 11. Tenmicroliters of human serum was spotted onto the second filter element.The device was inserted into a tube containing a 100 μl suspension ofcarbon particles (5 μl) labeled with flouresceinisothiocyanate-conjugated goat anti-human Immunoglobin G (see Example21) in 20 mM ethylenediaminetetraacetate. A detectable signal appears in5 minutes if the sample is seropositive.

EXAMPLE 13

[0102] An assay device was prepared according to Example 11 except 2mg/ml of Rubella antigen (Viral Antigens, Inc.) was line-sprayed on thewicking membrane.

[0103] When the reservoir pad is contacted with 100 μl of human serumand 5 μl carbon particles labeled with flourescein isothiocyanateconjugated goat anti-human Immunoglobin G (see Example 21), a detectablesignal appears in about 2 minutes if the sample is seropositive.

EXAMPLE 14

[0104] An assay device was prepared according to Example 13. Thenmicroliters of serum was spotted on the second filter element and thedevice was inserted into a tube containing a 100 μl suspension of carbonparticles labeled with flourescein isothiocyanate conjugated goatanti-human Immunoglobin G (see Example 21) in 20 mMethylenediaminetetraacetate.

[0105] A detectable signal appears in about 3 minutes if the sample isseropositive.

EXAMPLE 15

[0106] An assay device was prepared according to Example 11 and anadditional line of Rubella antigen was sprayed approximately 7 mm fromand parallel to the line of lyme antigen.

[0107] A Rubella seropositive sample (10 μl) was spotted on the secondfilter element. The device was inserted into a tube containing a 100 μlsuspension of carbon particles (10 μl) labeled with flouresceinisothiocyanate conjugated goat anti-human Immunoglobin G (see Example21) in 20 mM ethylenediamine tetraacetate.

[0108] A detectable signal appeared in about 5 minutes along the line ofRubella antigen.

EXAMPLE 16

[0109] The procedure in Example 15 was followed except that a Rubellaand Lyme seropositive sample (20 μl) was spotted on the membrane. Twodetectable signals began to appear in about 5 minutes.

EXAMPLE 17

[0110] The suitability of different carbon materials for preparation ofthe carbon sols and buffers for the same can be readily determined bythe following techniques.

[0111] A. Mixtures of 5 mg of different carbon black (Monarch 1,000,Monarch 880, Monarch 120, Regal 250R, Regal 500R, Vulcan XC72R, andVulcan XC72, all obtained from Cabot) and 100 μl of 2% polyethyleneglycol (6,000-8,000) are ground for 5 min. and diluted up to 10 ml withphosphate saline buffer containing 2 mg of a monoclonal antibody madeagainst human chorionic gonadotropin. After a brief sonication todisperse the carbon particles in the monoclonal antibody solution, themixtures are incubated for 6 hours at ambient temperature with stirring.At the end of the incubation, the sample is washed three times bycentrifugation to remove any excess antibody. Each centrifugation iscarried out at 15,000 RPM for 20 min. using 10 ml of phosphate buffersolution. The final pellet is suspended in 10 ml of 3% phosphate buffersolution and sonicated briefly to ensure complete dispersion of thecarbon particles.

[0112] For human chorionic gonadotropin assay, 20 μl of the carbon soland 200 μl of urine sample are dispersed and mixed well in a culturetube (10×75 mm) The mixture is then allowed to migrate into a strip ofWhatman paper (31ET) measuring 5 mm in width and 100 mm in height,line-sprayed with sheep anti-human chorionic gonadotropin antibody andblocked with 1% bovine serum albumin in phosphate buffer solution (pH7.4).

[0113] Vulcan XC72 appears to give the best signal-to-noise ratio at 200mIU/ml human chorionic gonadotropin. Similar results are obtained withVulcan XC72R, but the positive signal is slightly lower.

[0114] B. Five milligrams of the same carbon blacks are suspended in 2ml of 20 mM Tris-HCl buffer (pH 6.8) containing 40 mm sodium chlorideand 2% (w/v) dextran 9,400 by homogenization. After 2 hours ofincubation at ambient temperature, 1 ml of 3% bovine serum albuminsolution is added to the homogenized carbon suspension. The mixture issonicated briefly and incubated further for approximately 12 hours atambient temperature. At the end of the incubation, 5 μl of the mixtureis dispensed in a cuvette containing 1 ml of distilled water. Absorbencyat 700 nm is measured for each sample. The results are as follows:Source of Carbon Black OD at 700 nm Monarch 1,000 0.2333 Monarch 8800.3129 Vulcan XC72R 0.6878 Vulcan XC72 0.7428 Monarch 120 0.6225 Regal250R 0.3567 Regal 500R 0.4372

[0115] C. Vulcan XC72 carbon black is suspended in several buffersolutions having different pH. Five milligrams of Vulcan XC72 carbonparticles are homogenized in 2 ml of different buffer solutionscontaining 2% dextran 9,400 and incubated for 2 hours at ambienttemperature. After the incubation, 5 μl of each homogenate are added to1 ml of distilled water. One milliliter of 3% bovine serum albumin inthe same buffer is added to the mixture which is then sonicated andincubated for approximately 12 hours at ambient temperature. At the endof the incubation, 5 μl of the mixture are suspended in 1 ml ofdistilled water and absorbency is measured at 700 nm. The results are asfollows: OD at 700 nm Bovine Ionic Serum Buffers Strength pH DextranAlbumin sodium phosphate 0.1 M 6.0 0.3335 0.6030 sodium phosphate 0.1 M6.8 0.5142 0.7462 Tris-HCl 0.02 M 6.8 0.6277 0.8452 sodium phosphate0.003 M 7.0 0.4722 0.5389 sodium phosphate 0.1 M 7.6 0.4348 0.6100Tris-HCl 0.02 M 8.0 0.6479 0.5220 glycine-HCl 0.1 M 8.3 0.4666 0.5197Tris-citrate 0.1 M 8.6 0.4284 0.4933

[0116] As can be seen, buffer solutions having pH values of about 6.8 to8 are particularly good for the dispersion of carbon particles.

EXAMPLE 18

[0117] To a mixture of 1 mg of anti-human chorionic gonadotropinantibody in 1 ml of 0.3 M borate buffer (pH 9.0) are added with stirring50 μg of flourescein isothiocyanate. Stirring is continued for one hourand the mixture is then passed over a Sephadex G-25 column to removeunreacted isothiocyanate and other unwanted materials. The ratio ofantibody:isothiocyanate was approximately 1:3. To an aqueous suspensionof 1 mg of carbon black (Vulcan 72) is added 0.5 mg of the antibodyconjugate. The mixture is sonicated, incubated for about 12 hours atambient temperatures, and subjected to centrifugation three times. Thefinal pellet, suspended in a buffer such as described in Example 12, canbe stored at 4° C. until use.

[0118] Similar products can be obtained utilizing anti-luteinizinghormone, goat anti-human Immunoglobin G, and Immunoglobin M antibodies.

EXAMPLE 19

[0119] To 10 ml of an aqueous suspension of 5 mg of carbon black (Vulcan72) is added 200 μl of goat anti-mouse antiserum. The mixture issonicated and incubated for about 12 hours at ambient temperatures.There then is added 1 mg of anti-human chorionic gonadotropin antibodyand this mixture is incubated for two hours at ambient temperatures andsubjected to centrifugation three times. The final pellet, suspended ina buffer such as described in Example 12, can be stored at 4° C. untiluse.

EXAMPLE 20

[0120] To a suspension of 5 mg of carbon black in 10 ml of phosphatebuffer solution (PBS) are added 2 mg of avidin. After incubation for twohours at ambient temperature, 5 ml of 3% bovine serum albumin in PBS areadded. After standing for two hours, 0.5 mg of biotinylated anti-humanchorionic gonadotropin in 1% bovine serum albumin in PBS is added. Afteran additional one hour incubation, the mixture is subjected tocentrifugation three times. The final pellet, suspended in a buffer suchas described in Example 12 and then briefly sonicated, can be stored at4° C. until use.

EXAMPLE 21

[0121] Ten milligrams of Vulcan XC72 carbon particles are homogenized in2 ml of 20 mM Tris-hydrochloride buffer (pH 6.8) containing 40 mM sodiumchloride and 2% dextran 9,400. After 2 hours incubation at ambienttemperature; a solution of 5 mg of flourescein isothiocyanate in 1 ml ofTris-hydrochloride buffer is added to the solution. The mixture isbriefly sonicated and incubated for approximately 12 hours at ambienttemperature. After incubation, 20 ml of 0.1 M sodium phosphate buffer(pH 7.6) in 0.1 M sodium chloride are added to the carbon solution whichthen is centrifuged at 4° C. at 15,000 RPM. This step is repeated threetimes and the resultant pellet suspended in 20 ml of phosphate buffer.

[0122] Two milligrams of bovine serum albumin are added to 2 ml of theabove suspension and the mixture incubated for six hours and thensubjected to centrifugation three times. Excess glutaraldehyde (1%) isadded and after incubation for three hours at ambient temperatureremoved by centrifugation. A solution of 10 μg of thyroxine insufficient dimethylformamide is added and this mixture is incubated forthree hours at ambient temperature and then subjected to centrifugationthree times. The final pellet, suspended in a buffer such as describedin Example 12 and then briefly sonicated, can be stored at 4° C. untiluse.

What is claimed is:
 1. An immunochemical assay device comprising: a basemember; an array disposed on said base member, said array comprising:(i) a reservoir pad having sufficient porosity and volume to receive andcontain a liquid sample on which the assay is to be performed; (ii) awicking membrane disposed distally to said reservoir pad, said wickingmembrane having sufficient porosity and volume to absorb a substantialproportion of the sample received in said reservoir pad; and (iii) atleast one filter zone interposed between and contiguous with saidwicking membrane and said reservoir paid, said filter zone being (a)contiguous across a surface of said reservoir pad which is sufficientlysmall with respect to the volume of said reservoir pad to meter thepassage of the liquid sample from said reservoir pad to said filter zoneand (b) operable to permit passage of any specific ligand-receptorcomplex in said sample from said reservoir pad to said wicking membranewhile impeding passage of larger components then contained in saidsample; and at least one immobilized substance disposed in at least onezone of said wicking membrane and defining assay indicia, saidimmobilized substance being operable to bind a specific ligand-receptorcomplex contained in the sample to form said assay indicia.
 2. The assaydevice according to claim 1 in which the base member is plastic orglass.
 3. The assay device according to claim 1 in which said reservoirpad extends beyond said base member.
 4. The assay device according toclaim 1 in which at least one reagent operable to produce a specificligand-receptor complex is uniformly impregnated in and throughout atleast a portion of said filter zone.
 5. The assay device according toclaim 4 in which said reagent carries a label.
 6. The assay deviceaccording to claim 5 in which the label is a direct label.
 7. The assaydevice according to claim 6 in which the label is a metal sol, anonmetal sol, a dye sol, a color indicator, or particulate latex.
 8. Theassay device according to claim 7 in which the label is a carbon sol. 9.The assay device according to claim 6 in which the label is an indirectlabel.
 10. The assay device according to claim 4 in which said reagentand immobilized substance are ligand binding molecules.
 11. The assaydevice according to claim 4 in which at least one of said reagent andsaid immobilized substance is a monoclonal antibody.
 12. The assaydevice according to claim 4 in which at least one of said reagent andsaid immobilized substance is a polyclonal antibody.
 13. The assaydevice according to claim 1 in which said immobilized substance is aligand.
 14. The assay device according to claim 13 in which saidimmobilized substance is an antigen or hapten.
 15. The assay deviceaccording to claim 1 in which said filter zone comprises at least onefirst filter element disposed on said base member adjacent to saidreservoir pad and contiguous across a surface of said reservoir padsufficiently small with respect to the volume of said reservoir pad tometer the passage of the liquid sample from said reservoir pad to saidfirst filter element; and a second filter element disposed on said basemember adjacent to each first filter element and distal to saidreservoir pad, said second filter element being operable to permitpassage of any specific ligand-receptor complex in said sample butimpede passage of larger components then contained in said sample. 16.The assay device according to claim 15 in which at least one reagentoperable to produce a specific ligand-receptor complex is uniformlyimpregnated in and throughout at least a portion of said first filterelement.
 17. The assay device according to claim 16 in which each ofsaid pad, first filter element, second filter element, and wickingmembrane comprises microporous membrane material.
 18. The assay deviceaccording to claim 17 in which each microporous membrane comprisesnylon, cellulosic material, polysulfone, polyvinylidene difluoride, orpolyester.
 19. The assay device according to claim 1 in which anadditive operable to enhance the production or binding of specificligand-receptor complexes is present in said first filter element.
 20. Adevice according to claim 1 including a moisture impervious envelopesurrounding said device, said envelope including an aperture defined insaid envelope, the surrounding upper surface of which is curved andextends downwards forming a cup-like receptacle terminating and firmlyengaging a portion of said reservoir pad, said aperture being operableto receive and meter the passage of liquid sample onto said reservoirpad, and a means operable to permit visual inspection of said assayindicia zone disposed above said zone.
 21. An immunochemical labelcomprising finely particulate carbon black on which is adsorptivelyimmobilized a component which terminates distally from the point ofadsorption with an immunologically active ligand or ligand bindingmolecule.
 22. An immunochemical label according to claim 21 in whichsaid component consists of immunologically active haptens, antigens, orantibodies adsorbed on the surface of said carbon black.
 23. Animmunochemical label according to claim 21 in which said componentcomprises the immunological ligand or ligand binding moleculescovalently linked through a linking reagent and said linking reagent isadsorbed on the surface of said carbon black.
 24. An immunochemicallabel according to claim 23 wherein the linking reagent is an imide,azide, isothiocyanate, imidoester, or dialdehyde.
 25. An immunochemicallabel according to claim 24 wherein the linking reagent is maleimide,succinimide, phenylazide, glutaraldehyde, or N-hydroxysuccinimide ester.26. An immunochemical label according to claim 25 wherein the linkingreagent is an isothiocyanate.
 27. An immunochemical label according toclaim 26 wherein the isothiocyanate is phenylisothiocyanate,4,4′-diisothiocyanostilbene-2,2′-disulfonic acid,4-N,N-dimethylaminoazobenzene-4′-isothiocyanate, flouresceinisothiocyanate, or rhodamineisothiocyanate.
 28. An immunochemical labelaccording to claim 26 wherein the linking reagent isphenylisothiocyanate.
 29. An immunochemical label according to claim 26wherein the linking reagent is4,4′-diisothiocyanostilbene-2,2′-disulfonic acid.
 30. An immunochemicallabel according to claim 26 wherein the linking reagent is4-,N-dimethylaminoazobenzene-4′-isothiocyanate.
 31. An immunochemicallabel according to claim 26 wherein the isothiocyanate is flouresceinisothiocyanate.
 32. An immunochemical label according to claim 26wherein the linking reagent is rhodamineisothiocyanate.
 33. Animmunochemical label according to claim 21 wherein the finelyparticulate carbon black and immobilized component adsorptivelyimmobilized thereon are coated with polyethylene glycol having amolecular weight of from about 200 to about 20,000 or dextran having amolecular weight of from about 10,000 to about 500,000.
 34. Animmunochemical label according to claim 33 wherein the dextran has amolecular weight of from about 10,000 to about 50,000.
 35. Animmunochemical label according to claim 33 wherein the polyethyleneglycol has a molecular weight of from about 5,000 to about 12,000. 36.An immunochemical label according to claim 21 wherein said componentcomprises the immunochemical ligand or ligand binding molecules bound toa protein and said protein is adsorbed on the surface of said carbonblack.
 37. An immunochemical label according to claim 36 wherein theimmunological ligand or ligand binding molecules covalently linked tosaid protein through an immunological bond.
 38. An immunochemical labelaccording to claim 36 wherein the immunological ligand or ligand bindingmolecules covalently linked to said protein through a linking reagent.39. An immunochemical label according to claim 21 wherein said componentcomprises the immunological ligand or ligand binding molecules bound toa protein, said protein is covalently linked to a linking reagent andsaid linking reagent is adsorbed on the surface of said carbon black.40. An immunochemical label according to claim 39 wherein theimmunological ligand or ligand binding molecules covalently linked tosaid protein through a second linking reagent.
 41. An aqueous suspensionof an immunochemical label according to claim
 21. 42. An aqueoussuspension according to claim 41 including at least one buffer providinga pH at which the immobilized immunological ligand is stable and fallingwithin the range of from about 6 to
 9. 43. An aqueous suspensionaccording to claim 42 including at least one buffer providing a pH offrom about 6.5 to about 8.5.
 44. The method of preparing animmunochemical label according to claim 23 which comprises linkingimmunological ligand or ligand binding molecules to the finelyparticulate carbon black by, simultaneously or sequentially, allowing alinking reagent to both (i) react covalently with the immunologicalligand or ligand binding molecules and (ii) be adsorbed on finelyparticulate carbon black.
 45. The method according to claim 44 whereinthe immunochemical label is contacted with an aqueous solution of apolyethylene glycol having a molecular weight of from about 100 to about20,000.
 46. The method according to claim 44 wherein the finelyparticulate carbon black is contacted with an aqueous solution of adextran having a molecular weight of from about 10,000 to about 500,000.47. The method according to claim 44 wherein the immunological ligand orligand binding molecules are linked to the finely particulate carbonblack through an imide, azide, isothiocyanate, imidoester, ordialdehyde.
 48. The method according to claim 44 wherein theimmunological ligand or ligand binding molecules are linked to thefinely particulate carbon black through an isothiocyanate.
 49. Themethod according to claim 44 wherein the immunological ligand or ligandbinding molecules are linked to the finely particulate carbon blackthrough phenylisothiocyanate,4,4′-diisothiocyanostilbene-2,2′-disulfonic acid,4-N,N-dimethylaminoazobenzene-4′-isothiocyanate, flouresceinisothiocyanate, or rhodamineisothiocyanate.
 50. The method according toclaim 44 wherein the linked immunological ligand or ligand bindingmolecules and finely particulate carbon black are suspended in anaqueous medium buffered to a pH falling with the range of from about 6to about 9 and at which the immobilized immunological ligand or ligandbinding molecule is stable.
 51. The method according to claim 50 whereinthe aqueous suspension is treated with at least one biologicallyacceptable ionic or nonionic surfactant.
 52. In an immunochemical testdevice utilizing a reaction between immunological ligand or ligandbinding molecules and an analyte, the improvement which comprises usingan immunochemical label comprising finely particulate carbon black onwhich is adsorptively immobilized a component which terminates distallyfrom the point of adsorption with an immunologically active ligand orligand binding molecule of said reaction.
 53. A device according toclaim 1 including a moisture impervious envelope surrounding saiddevice, said envelope including a first aperture defined in saidenvelope, the surrounding upper surface of which is curved and extendsdownwards forming a cup-like receptacle terminating and firmly engaginga portion of said reservoir pad, said first aperture being operable toreceive and meter the passage of liquid onto said reservoir pad, asecond aperture defined in said envelope directly above said secondfilter element, said second aperture being operable to permit directapplication of sample onto said second filter element, and a meansoperable to permit visual inspection of said assay indicia zone disposedabove said zone.